The broad, long-term objective of this proposal is to study several mouse models of human disease, growth and development with innovative technology to better understand the role of glycolipids and sphingolipids in mental retardation, and to explain mechanisms of therapeutic reversal. The expected outcome of our two major specific aims, is to verify the hypothesis that microdomain regulation of ceramide, sphingosine and sphingosine-1-phosphate (S1P) is achieved by several different mechanisms, that S1P depletion is common to different neuropathological states and sphingoid-mimicking drugs can play an important role in reversing mental retardation Aim 1. We will take advantage of our recent discovery that the deletion of the gene for NSMase 2 in mice reveals a role for glycolipids, ceramide and SM in brain growth and skeletal development through regulation of AktP and the mTor pathway, creating a model for human osteogenesis imperfecta. We will use a second mouse model (ASMase (-/-)) to further show how the two major SMases are co-regulated and the role of lysosomal hydrolases in the regulation of the Cer/S1P ratio. To achieve this goal we will use HPLC/MS/MS lipidomics and take advantage of the ability of sphingolipids to form microdomains to assemble signaling complexes which can be visualized by transfecting cells with GFP-GPI anchored protein constructs. Aim 2. We will use a drug-based approach and in vivo animal models, expanding on our recent observation that FTY720 (approved for therapy of Multiple Sclerosis) is lipophilic cationic drug and a functional inhibitor of acid sphingomyelinase (FIASMA) with functional consequences for glycolipid, SM, ceramide and S1P metabolism. To better understand S1P regulation and function we will use an in vivo remyelination model (cuprizone-treated mouse) to determine the effects of FTY720 in vivo and the epigenetic effects of S1P on gene transcription through inhibition of histone deacetylases (HDAC 1/2). We will use MS/MS proteomics to identify specific protein acylation patterns under different Cer/S1P ratios and how this relates to remyelination. We will compare these results with the effect of a glucosyltransferase inhibitor drug (EtDoP4/Epiglustat), in which substrate reduction reverses the depletion of Ceramide and S1P in a number of lysosomal storage diseases involving mental retardation as well as AIDs and Parkinsons disease. The impact of this research will be to better understand how sphingolipids work so that combination drug therapies can be used to treat patients in the future.